Intro To HPLC
Intro to HPLC
It’s time to learn what HPLC is.
HPLC is useful for many things such as identifying and separating components to know what exactly is in each substance
So let’s get started.
What is HPLC?
HPLC stands for High pressure liquid chromatography.
It is used to separate liquid components into different layers for quantitative and qualitative analysis.
It originated from a Russian botanist named Mikhail Semenovich Tswett in the 1900s. What he did was pack some Calcium Carbonate in a standing tube and added pigments extracted from plants before flushing it with petroleum where it separated into multiple layers of colors. Hence the name chroma (which means color) in Greek.
How Does The Separation Mechanism Work?
In HPLC, there is something called the stationary phase and the mobile phase.
In other words, the liquid solution that’s moving through the HPLC is called the mobile phase and the stationary phase is a column that stays still where the liquid solution passes through.
In other words, we are separating components by letting the liquid solution pass through the stationary phase (ex. column) and then analyzing it in a computer.
Now the way it separates it gets a little bit more complex. For our example we are going to talk about the most commonly used HPLC technique called the reverse-phase HPLC which uses a polar mobile phase and a nonpolar column.
In science, like attracts like. This means that a polar is strongly attracted to another polar molecule and does not have much attraction to something that is nonpolar.
So when you pour your samples through the HPLC, that means the nonpolar is going to stick to the nonpolar column and the polar molecules that don’t have much attraction will go straight through.
This is how it separates where in this case the polar molecules go out first because there isn’t much attraction whereas with the nonpolar molecules in the sample they are going to come out slower because they are being attracted by the nonpolar molecules.
Think about it like this. Imagine a tiny water slide with people coming out of the same slide at different speeds. The tiny and skinny people who can go through the slide will come out faster because they are maximized to not touch the slide making them go faster while the bigger people may be slower because the slides are too small making it cramp to gain acceleration.
To sum it up in a nonpolar column (nonpolar stationary phase) with a polar solvent (polar mobile phase):
- The polar molecules will come out first since there’s less attraction.
- The nonpolar molecules will come out later because there’s more attraction.